Xylase isomerase purified from thermotoga maritima and thermotoga neapolitana

ABSTRACT

This invention is in the field of glucose isomerization enzymes. More specifically, the invention is directed to a novel xylose isomerase, a process for the preparation of this enzyme, the use of this enzyme in glucose isomerization processes, and glucose isomerization processes. The enzyme is preferably derived from Thermotoga maritima or Thermotoga neapolitana. The enzyme has a temperature optimum above 90° C., pH optimum in the range of from 6 to 7 and a residual activity at 90° C. of more than 40% after 30 minutes and/or residual activity at 98° C. of more than 20% after 30 minutes. The enzyme can also be in immobilized form.

TECHNICAL FIELD

This invention is in the field of glucose isomerization enzymes. Morespecifically, the invention is directed to a novel xylose isomerase, aprocess for the preparation of this enzyme, the use of this enzyme inglucose isomerization processes, and glucose isomerization processes.

BACKGROUND ART

Glucose isomerization enzymes catalyze the conversion of glucose(dextrose) to fructose in the manufacture of High Fructose Syrup (HFS),i.e. a syrup containing a mixture of glucose and fructose. All describedglucose isomerases fundamentally are xylose isomerases. Thus, for thesake of clarity, the enzyme of this invention ordinarily will beidentified as xylose isomerase, although the most important applicationthereof is for the isomerization of glucose to fructose.

Due to the high sweetening power of fructose and the low tendency tocrystallize HFS is widely used in industry, where it can replace liquidsucrose and invert syrup in many food and beverage products such assoft-drinks, baked goods, canned fruit, ice cream, confectionery, jamsand jellies.

A large number of patents are directed to different microbial sources ofxylose isomerase. Many bacteria, e.g. species belonging to the generaStreptomyces; Actinoplanes; Bacillus: and Flavobacterium, and fungi,e.g. species belonging to the class Bacidiomycetes, have been identifiedin the patent literature as xylose isomerase producing microorganisms,vide e.g. U.S. patent publication No. 4,687,742 and EP patentpublication No. 352,474.

For many years the wish has existed to perform the isomerization processat elevated temperatures, vide e.g. U.S. patent publication Nos.4,410,627; 4,411,996; and 4,567,142. The conversion of glucose tofructose is a temperature dependent equilibrium reaction: The higher thetemperature, the higher the yield of fructose at equilibrium. However,the isomerization temperature strongly affects the enzyme activity andstability. Higher temperatures will result in higher enzyme activity(increased glucose conversion rate), but also increased thermaldenaturation of the enzyme. Therefore, the stability, and hence theproductivity, decreases with higher temperatures. On the other hand,lower isomerization temperatures will lead to increased risk ofmicrobial infection. The infection risk is minimized when operating attemperatures of from approximately 60° C.

Of outstanding commercial interest are syrups containing approximately55% (w/w dry substance) fructose. At this fructose level the syrupattains equal sweetness with sucrose on a weight to weight dry basis,and is used interchangeably as a total or partial replacement forsucrose in many food products, and especially in carbonated soft drinks.

Regards to thermal stability and enzyme consumption greatly influencethe isomerization conditions. Thus, in practice, due to the life time ofthe enzyme and the productivity, syrups containing more thanapproximately 42% fructose are obtained by non-enzymatic treatment ofthe enzymatically manufacture HFS. The non-enzymatical treatmentcomprises expensive chromatographical separation to obtain a syrup withthe desired fructose concentration. Therefore, an extensive search forxylose isomerases that allow the isomerization process to be carried outat higher temperatures in favour of a higher fructose yield has takenplace.

It is an object of the invention to provide a xylose isomerase that inrespect to thermal stability is superior over any hitherto known xyloseisomerase. Moreover, it is an object of the invention to provide aisomerization process for high yield glucose conversion.

SUMMARY OF THE INVENTION

Now a novel xylose isomerase has been found that possesses outstandingglucose isomerization properties. Accordingly, in its first aspect, thepresent invention provides a xylose isomerase having a temperatureoptimum above 90° C.; pH optimum in the range of from pH 6 to 7; aresidual activity after 30 minutes at 90° C. of more than 40%, or aresidual activity after 30 minutes at 98° C. of more than 20%. Inanother aspect, the present invention provides a xylose isomerase havinga temperature optimum above 90° C.; pH optimum in the range of from pH 6to 7; a residual activity after 30 minutes at 90° C. of more than 40%,and a residual activity after 30 minutes at 98° C. of more than 20%; andhaving immunochemical properties identical or partially identical tothose of the xylose isomerase derived from Thermotoga maritima. DSM No.3109, or Thermotoga neapolitana. DSM No. 4359 or DSM No. 5068. In a morespecific aspect of the present invention, there is provided a xyloseisomerase obtainable from a xylose isomerase producing strain ofThermotoga sp.; preferably T. maritima or T. neapolitana. In a yet morespecific aspect of the present invention, there is provided a xyloseisomerase obtainable from T. maritima. DSM No. 3109, or T. neapolitana,DSM No. 4359 or DSM No. 5068, or a mutant or a variant thereof. In apreferred embodiment of the present invention, there is provided axylose isomerase in the form of an immobilized enzyme.

In a third aspect of the invention, there is provided a process for thepreparation of the enzyme, which process comprises cultivation of axylose isomerase producing strain of Thermotoga sp. in a suitablenutrient medium, containing carbon and nitrogen sources and inorganicsalts, followed by recovery of the desired enzyme. In a more specificaspect, a strain of T. neapolitana or T. maritima is cultivated. In ayet more specific aspect, T. maritima. DSM No. 3109, or T. neapolitana,DSM No. 4359 or DSM 5068, a mutant or a variant thereof, is cultivated.

In a fourth aspect of the invention, the use of a xylose isomerase ofthe invention in glucose isomerization processes is claimed. In a morespecific aspect, there is provided a glucose isomerization process, inwhich a feed liquor, containing from about 20 to 65% (w/w dry substance)glucose, is reacted with a xylose isomerase of the invention at atemperature of from 50° to 130° C., at a pH of from 3.5 to 8, and areaction time of from 10 seconds to about 5 hours. In another specificaspect, there is provided a two step glucose isomerization process, inwhich a feed liquor, containing from about 20 to 65% (w/w dry substance)glucose, in the first step is reacted with an isomerization enzyme at atemperature of from 50° to 80° C., at a pH of from 6 to 8, and areaction time of from 10 minutes to about 5 hours, to produce a highfructose syrup, containing from about 40 to about 50% fructose, whichhigh fructose syrup in the second step is reacted with an isomerizationenzyme at a temperature of from 80° to 130° C., at a pH of from 3.5 to8, and a reaction time of from 10 seconds to about 5 hours, to produce ahigh fructose syrup, containing from about 50 to about 60% fructose, theisomerization enzyme employed in the second step being a xyloseisomerase of the invention. In a preferred embodiment, the isomerizationenzyme employed in both steps is a xylose isomerase of the invention. Inanother preferred embodiment, the xylose isomerase is immobilized andretained in a fixed bed, through which the glucose or glucose/fructosesolution flows.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to theaccompanying drawings, in which:

FIG. 1 shows the relation between temperature and activity of the enzymeof the invention;

FIG. 2 shows the relation between pH and activity of the enzyme of theinvention;

FIGS. 3A, 3B, 4A and 4B show a comparison of the residual activity(thermostability) of the enzyme of the invention and a known xyloseisomerase at various temperatures (70°, 80°, 90° and 98° C.);

FIG. 5-6 show the effect of divalent metal ions; and

FIG. 7 shows the relation between cell density (cells/ml) and OD₆₀₀,respectively, and the fermentation time (hours).

DETAILED DISCLOSURE OF THE INVENTION The Enzyme

The enzyme of the invention is a novel xylose isomerase that catalyzesthe conversion of D-glucose to D-fructose. The enzyme possessesthermostability superior to any known xylose isomerase. Theextraordinary thermostability is demonstrated by comparison to an enzymerepresentative of the thermostability of the known xylose isomerases,namely a xylose isomerase obtained from Streptomyces murinus (vide U.S.patent publication No. 4,687,742 and EP patent publication No. 352,474).The xylose isomerase of the invention can be described by the followingcharacteristics.

Physical-Chemical Properties

As shown in FIG. 1, the xylose isomerase of the invention is active in atemperature range of from below 60° C. to above 98° C. The temperatureoptimum is at temperatures above 90° C., more precisely above 95° C.

As shown in FIG. 2, the enzyme of the invention has pH optimum in therange of from pH 6 to 7.5, more precisely pH 6 to 7 (around pH 6.6),when measured in a MOPS (3-(N-mor-pholino)-propanesulphonic acid) buffersystem (cf. Example 3).

As shown in FIGS. 3-4, the thermostability of the enzyme of theinvention, described as the residual activity, is:

At 70° C.: More than 60%, preferably more than 80%, after 5 hours; ormore than 30%, preferably more than 50%, after 33 hours.

At 80° C.: More than 60%, preferably more than 80%, after 3 hours; ormore than 30%, preferably more than 50%, after 24 hours.

At 90° C.: More than 40%, preferably more than 60%, more preferably morethan 80%, after 30 minutes; or more than 20%, preferably more than 40%,more preferably more than 60%, after 6 hours.

At 98° C.: More than 20%, preferably more than 40%, more preferably morethan 60%, yet more preferably more than 80%, after 30 minutes; or morethan 20%, preferably more than 40%, after 6 hours.

Investigations in relation to the effect of metal ions on the enzyme ofthe invention showed a stabilizing effect of Co²⁺ and Mg²⁺. This effectis illustrated in FIGS. 5-6.

Immunochemical Properties

The immunochemical properties can be determinated immunologically bycross-reaction identity tests. The identity tests can be performed bythe well-known Ouchterlony double immunodiffusion procedure or by tandemcrossed immunoelectrophoresis according to N. H. Axelsen; Handbook ofImmunoprecipitation-in-Gel Techniques; Blackwell Scientific Publications(1983), chapters 5 and 14. The terms "antigenic identity" and "partialantigenic identity" are described in the same book, chapters 5, 19 and20.

Monospecific antiserum is generated, according to the above mentionedmethod, by immunizing rabbits with the purified xylose isomerase of theinvention. The immunogen is mixed with Freund's adjuvant and injectedsubcutaneously into rabbits every second week. Antiserum is obtainedafter a total immunization period of 8 weeks, and immunoglobulin isprepared therefrom as described by N. H. Axelsen, supra.

Preparation of the Enzyme

The enzyme is obtainable by cultivation of a xylose isomerase producingstrain of Thermotoga sp. in a suitable nutrient medium, containingcarbon and nitrogen sources and inorganic salts, followed by recoveryand purification of the desired enzyme by conventional means. The enzymecan also be obtained by recombinant DNA-technology.

A process illustrating the preparation of the enzyme of the invention byfermentation is described in Example 1.

The Microorganism

The enzyme of the invention is obtainable from thermophilic eubacteriabelonging to the genus Thermotoga (Thermotogales).

The organisms belonging to the genus Thermotoga grow at temperatures offrom 55° to 90° C., with growth optimum around 80° C. They are obligateanaerobes (somewhat O₂ tolerant). They are halotolerant, and growoptimally at 2.7% NaCl, with a range of 0.25 to 3.75% NaCl. They growoptimally at pH 6.5, with a pH range of 5.5 to 9. They are obligateheterotroph, show fermentative growth on a range of saccharides,including glucose, ribose, xylan, maltose, starch, etc. They can grow inpresence or absence of S^(o), and produce H₂ (H₂ S), CO₂, acetate,lactate, and several other minor organic products.

Three strains of the genus Thermotoga are deposited: T. maritima, DSMNo. 3109; and T. neapolitana, DSM No. 4359 and DSM No. 5068.

The Isomerization Process

By employing the enzyme of the invention in an isomerization process,the reaction temperature -- and hence the yield of fructose -- can beelevated, when compared to hitherto known isomerization processes. In aone step process of the invention the temperature should be in the rangeof from 50° to 130° C.

In another aspect of the invention, a two step isomerization process iscarried out. In this process, the isomerization, in its first step, iscarried out as a conventional isomerization process, employing either aconventional isomerization enzyme or a xylose isomerase of theinvention, to produce a high fructose syrup containing from about 40 to50% fructose. In the second step, the end product from the first step issubjected to isomerization at elevated temperatures, i.e. of from 80° to130° C., and at a pH of from 3.5 to 8, by employing a xylose isomeraseof the invention, to produce a high fructose syrup containing from about50 to 60% fructose. The rationale behind this process being that thetime necessary for the product to stay at elevated temperatures (thesecond process step) can be reduced considerably, when employing astarting product that is already a high fructose syrup.

Other isomerization conditions can be as for conventional isomerizationprocesses.

The reaction time can be in the range of 10 seconds to 5 hours,depending on the isomerization temperature, specific activity of theenzyme preparation employed, etc.

The isomerization pH affects enzyme activity, stability and by-productformation. The isomerization pH should be in the range of from 3.5 to 8,more preferred 4.5 to 7. By-product formation due to glucose andfructose decomposition increases at higher pH levels.

The feed syrup dry substance content (DS) influences the rate offructose formation. Too high a DS level results in lower apparent enzymeactivity. On the other hand, too dilute a syrup will lead to a loweroptimum substrate/enzyme ratio and increased risk of microbialinfection. The feed liquor should contain of from 20 to 65%, morepreferred 30 to 60% w/w dry substance glucose.

The concentration of monosaccharides in the feed syrup should be as highas possible in order to obtain the maximum isomerization rate. With alow monosaccharide concentration in the feed syrup the isomerizationtemperature must be elevated in order to attain a given fructoseconcentration. For optimal performance of the isomerization process, thexylose isomerase of the invention can be immobilized. The isomerizationprocess of the invention can then be carried out as a continuous,fixed-bed reactor process. In addition to the convenience of continuousoperation, the fixed-bed process permits a short reaction time therebyminimizing by-product formation.

The enzyme can be immobilized by methods known in the art to producexylose isomerase preparations with acceptable high unit activities.

The following examples further illustrate the present invention.

EXAMPLE 1 Preparation

T. maritima, DSM No. 3109, was grown anaerobically in a 50 l fermentoron a medium with the following composition:

    ______________________________________                                        NaCl                  15.00  g/1                                              Na.sub.2 SO.sub.4     2.00   g/1                                              KCl                   0.35   g/1                                              NaHCO.sub.3           0.10   g/1                                              KBr                   0.05   g/1                                              H.sub.3 BO.sub.3      0.02   g/1                                              MgCl.sub.2 ; 6 H.sub.2 O                                                                            5.40   g/1                                              CaCl.sub.2 ; 2 H.sub.2 O                                                                            0.75   g/1                                              SrCl.sub.2 ; 6 H.sub.2 O                                                                            0.01   g/1                                              Tryptone (Difco)      3.0    g/1                                              Yeast Extract (Difco) 1.0    g/1                                              Xylose (Sigma X1500)  5.0    g/1                                              ______________________________________                                    

Xylose was sterilized separately. pH was adjusted to 6.0-6.5 with H₂ SO₄at room temperature. Na₂ S; 9 H₂ O (0.5 g/l) was added beforeinoculation. The fermentation was run at 80° C., 100 rpm, with an N₂sparge of about 0.05 vvm. An 8% inoculum was used (on the same media),and the run lasted about 8 hours, to an OD₆₀₀ of about 0.3. In FIG. 7the relation between cell density and optical density at 600 nM (OD₆₀₀),respectively, and the fermentation time is shown.

Cells were harvested using a Sharples centrifuge, and frozen untilsonification. The cell yield was 25 g wet weight.

Cell extracts were prepared by sonicating a cell suspension in a Tekmarsonicator for 6×30 seconds at 50% duty cycle. Membranes and cell debriswere removed by centrifugation at 25,000×g for 30 minutes. The extractwas dialyzed overnight against 50 mM MOPS buffer, pH 7.0, containing0.02% sodium azide, and stored at 4° C.

EXAMPLE 2 Analytical Method

Assay for xylose isomerase activity was performed the following way:50-200 μl cell extract, obtained as described in Example 1, were addedto 2.5 ml of a mixture containing 1 M fructose; 80 mM MOPS, pH 6.9; and10 mM Co²⁺. The mixture was incubated at 80° C. for 60 minutes, andglucose was determined by a standard glucose oxidase type assay (Sigma510).

EXAMPLE 3 Characterization

Temperature optimum: 50-200 μl cell extract, obtained as described inexample 1, were added to 2.5 ml of a mixture containing 1 M fructose; 80mM MOPS, pH 6.9; and 10 mM Co²⁺. Several such mixtures were incubatedfor 60 minutes at various temperatures in the range of from 60° C. to98° C. The result is shown in FIG. 1.

pH optimum: 50-200 μl cell extract, obtained as described in example 1,were added to 2.5 ml of a mixture containing 1M fructose and 10 mM Co²⁺,in each of the following buffer systems (pH values indicated on thefigure):

80 mM acetate;

80 mM MES;

80 mM MOPS; and

80 mM phosphate.

Each mixture was incubated for 60 minutes at 80° C., and assayed forglucose. The result is shown in FIG. 2.

Thermostability: The thermostability of the xylose isomerase obtainedfrom T. maritima was compared to the known thermostable xylose isomeraseobtained from Streptomyces murinus, by the following methods.

Xylose isomerase of the invention: 50-200 μl cell extract, obtained asdescribed in Example 1, were added to 2.5 ml of a mixture containing 1Mfructose; 80 mM MOPS, pH 6.9; and 10 mM Co²⁺. Several such mixtures wereincubated for 60 minutes at temperatures of 70°, 80°, 90° and 98° C.,respectively. Samples were taken out at various incubation times andassayed for glucose.

Xylose isomerase obtained from S. murinus: Purified xylose isomerase,prepared according to U.S. patent publication No. 4,687,742, was dilutedwith 50 mM MOPS buffer, pH 7.5, to a dry substance content of 4%. 50-200μL enzyme solution were added to 2.5 ml of a mixture containing 1Mfructose; 80 mM MOPS, pH 7.5; and 10 mM Co²⁺. Several mixtures wereincubated for 30 minutes at temperatures of 70°, 80°, 90° and 98° C.,respectively. Samples were taken out at various incubation times andassayed for glucose.

The results are shown in FIGS. 3-4.

Effect of divalent metal ions: 50-200 μl cell extract, obtained asdescribed in example 1, were added to 2.5 ml of a mixture containing 1Mfructose; 80 mM MOPS, pH 6.9; and one or more of the following metalions in the concentration cited:

Co²⁺, 10 mM;

Mg²⁺, 100 mM;

Mn²⁺, 10 mM;

Co²⁺ /Mg²⁺, 10 mM/50 mM;

Co²⁺ /Ca²⁺, 10 mM/2 mM; and

Co²⁺ /Ca²⁺ /Mg²⁺, 10 mM/2 mM/50 mM.

The mixtures were incubated for 60 minutes at 80° C., and assayed forglucose. The results are shown in FIGS. 5-6.

We claim:
 1. A substantially purified xylose isomerase which is derivedfrom Thermotoga maritima or Thermotoga neapolitana and has the followingproperties:(a) temperature optimum above 90° C.; (b) pH optimum in therange of from 6 to 7; and (c) a residual activity at 90° C. of more than40% after 30 minutes and/or a residual activity at 98° C. of more than20% after 30 minutes.
 2. The xylose isomerase according to claim 1 whichhas immunochemical properties identical or partially identical to thoseof the xylose isomerase derived from Thermotoga maritima DSM No. 3109,or Thermotoga neapolitana, DSM No.
 4359. 3. The xylose isomeraseaccording to claim 1, in the form of an immobilized enzyme.
 4. Thexylose isomerase according to claim 1, which is derived from Thermotogamaritima DSM No. 3109 or a mutant thereof.
 5. The xylose isomeraseaccording to claim 1, which is derived from Thermotoga neapolitana DSMNo. 4359 or a mutant thereof.